Array assays between surface bound binding agents or probes and target molecules in solution are used to detect the presence of particular biopolymers. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules in solution. Such binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.
One typical array assay method involves biopolymeric probes immobilized in an array on a substrate such as a glass substrate or the like. A solution containing analytes that bind with the attached probes is placed in contact with the array substrate, covered with another substrate such as a coverslip or the like to form an assay area and placed in an environmentally controlled chamber such as an incubator or the like. Usually, the targets in the solution bind to the complementary probes on the substrate to form a binding complex. The pattern of binding by target molecules to biopolymer probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample. In most instances, the target molecules are labeled with a detectable tag such as a fluorescent tag or chemiluminescent tag. The resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used. For example, laser light may be used to excite fluorescent tags, generating a signal only in those spots on the biochip that have a target molecule and thus a fluorescent tag bound to a probe molecule. This pattern may then be digitally scanned for computer analysis.
As such, optical scanners play an important role in many array based applications. Optical scanners act like a large field fluorescence microscope in which the fluorescent pattern caused by binding of labeled molecules on the array surface is scanned. In this way, a laser induced fluorescence scanner provides for analyzing large numbers of different target molecules of interest, e.g., genes/mutations/alleles, in a biological sample.
In performing scans, a typical approach is to zigzag across an array substrate obtaining data in a raster fashion. In doing so, it has been appreciated that very slight variation in the tilt or angle of a substrate being scanned must be accounted for in order to achieve acceptable focus on successive features to accurately obtain data. As variations in substrates and scanners which cause slight variations of the tilt or angle of a substrate are extremely common, a great need exists for scanners that can automatically correct for the variation, and can effectively focus and collect data across an entire substrate without user intervention. As such, an effective and robust “autofocus” scanner would be extremely attractive.
In general, focus is achieved in an optical scanning device using a system that actuates a scanning lens assembly or the cradle carrying a sample by a servomechanism by varying the distance between these two components. The most common types of electronic feedback logic controllers for effecting automatic light source actuation and focus presently in use are the Proportional-Integral (PI) and Proportional-Integral-Derivative (PID) controllers which analyze signals from several scanned position on a substrate, make a prediction of the focal length required for correct focus of a future position, and adjust the focus of a light source accordingly. The implementation of each of these controllers varies widely and tuning and custom design of each type are well within the ability of those with ordinary skill in the art.
PI or PID autofocus controllers and the like are, in theory, capable of automatically focusing a light source during scanning of an array substrate. However, a problem with all PI, PID and related focus controllers is that high frequency (150–1000 Hz) noise arising from bearing noise, position sensing detector (PSD) noise, stray light, resonances within the control system, variations in optical path arising from scan lens motion or other sources generate instabilities in the autofocus control. As such, the noise reduce the effectiveness of the controller and prevent array substrates from being held in focus while they are being scanned. Several solutions to this problem have been investigated.
One solution to the problem is known as the “feed forward” controller, which creates a mathematical model of the slide surface, and controls the focus indirectly, by controlling the absolute position of a substrate to a setpoint derived from the model. Such an approach works best when the system being controlled is precisely known, and the external perturbations to which it is subjected can be accurately modeled. A pure feed-forward controller cannot easily cope with the variety of different substrates and differences among scanners that are commonly encountered.
Another solution is to apply either an analog or digital low-pass or bandpass asymmetric filter to the focus error signal, so as to average out the higher frequency perturbations. However, digital and analog filters that are asymmetric, i.e. those which operate using inputs from earlier time points, introduce delays and phase shifts into the filtered data, which may destabilize an autofocus control loop. Furthermore, it is known that a 250 Hz low-pass asymmetric digital filter destabilizes the autofocus controller of at least one scanner, as do similar analog filters.
A further solution is to detune the autofocus control loop (i.e. reduce the gain of the control loop) so that it does not attempt to control out high-frequency noise. However, de-tuning severely degrades performance. Although many adaptive control methods are available that have the ability to detune an autofocus control loop, they are either excessively computationally intensive and/or insufficiently robust. As such, they tend to be impractical.
Accordingly, a need still exists for a scanner that automatically and effectively maintains focus of a light source during scanning of an array sample. The present invention meets this, and other, needs.
Relevant Literature
U. S. patents of interest include: U.S. Pat. Nos. 5,091,652; 5,260,578; 5,296,700; 5,324,633; 5,585,639; 5,760,951; 5,763,870; 6,084,991; 6,222,664; 6,284,465; 6,371,370 6,320,196 and 6,355,934.